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BBa_K1152005 Review 1 Not understood Username

Group: iGEM Team Marburg 2014 Author: iGEM Team Marburg 2014 Summary: The problem of previous applications such as the originally intended one is that the size of synthesized peptides is limited to 50 amino acids (Caboche et al., 2008). Therefore we established a system that uses the capabilities of NRPSs to introduce non-proteinogenic amino acids but is able to overcome the size limit of the synthesized peptides. By the fusion of the Phenylalanine activating module from this NRPS to a tRNA binding domain the catalytic activity of this NRPS module was introduced to the ribosomal pathway which has much more powerful capabilities regarding the protein synthesis as it can assemble huge proteins using RNA as a blueprint (http://2014.igem.org/Team:Marburg:Project:NRPS#improve). Purpose of our improvement Our aim was to make it possible to combine the advantages of the nonribosomal peptide synthesis with the ribosomal pathway, i.e. the enormous repertoire of amino acids combined with the ability of the ribosome to synthesize huge proteins. To reach that aim, we planed to create a fusion protein that has the capability to activate amino acids derived from NRPSs´ A-domain PheA and tRNA binding capabilities derived from a part of multi aaRSs complexes Arc1p-C. Results of the aminoacylation level measurement Measurements showed that all fusion constructs were able to load L- as well as D-phenylalanine onto tRNAPhe (a). The varying linker length showed a clear influence on the yield levels with the 2x-GSSG linker showing the highest catalytic activity yielding 11% loaded tRNA after 30 min while the 8x-construct reached only a maximum of 3% loaded tRNA as well as the linker-less version. The remaining constructs showed intermediate results. Furthermore the linking of the two domains leading to the increase in reactant concentration and the correct spatial arrangement is indeed important for the catalytic effect to occur since a mixture of the unlinked domains showed only background levels of aminoacylation (b). Further negative controls included testing the reaction without enzyme or ATP. As a positive control to evaluate the method phenylalanyl-tRNA synthetase (PheRS) was used. A time-dependent measurement of the aminoacylation level showed that a maximum is reached after 30 min (c). To test if other tRNAs except for the tRNAPhe can be aminoacylated using our fusion construct we carried out aminoacylation assays with five additional E. coli tRNAs (d). The measurements suggest in agreement with previous studies that all tRNAs were loaded similarly well. Further information: see our project description and BBa_K1329005 Measurement method of aminoacylation levels tRNA, amino acid and the fusion protein were incubated and the reaction started by the addition of ATP. The reaction was stopped by the addition of sodium acetate, tRNA was precipitated with ethanol and purified by size exclusion chromatography. Half of the sample was treated with base, reacidified and analysed by LCMS.

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